SPIM is a fluorescence microscopy imaging technique that enables extended imaging of live dynamic samples due to significant reduction in phototoxicity and photo-bleaching compared to more conventional microscopy techniques. SPIM employs lines or planes of excitation illumination (often called light sheets), for example generated by a cylindrical lens, and perpendicular detection geometry to allow optically sectioned examination of samples. In addition, SPIM illumination generates a reduced out of focus background signal which greatly enhances the image quality of three-dimensional specimens. An example of SPIM is shown in U.S. Pat. No. 7,554,725.
In most implementations of SPIM, the SPIM system is designed around the specimen, thereby requiring novel sample preparation, such as embedding the sample in an agarose gel, which precludes the use of conventional sample mounts, such as glass cover slips, that are used with many conventional microscopes. Although other types of microscopy, such as oblique plane microscopy (OPM) use the same objective to illuminate as well as detect the specimen and can accommodate the above novel sample preparation, such a microscopy system suffers from a low numerical aperture relative to other methodologies, and therefore requires extensive optics to correct aberrations that result from the light sheet being tilted relative to the detection plane of the microscope. Consequently, it would be desirable to be able to easily convert an existing conventional microscope to accommodate SPIM. Such a conversion is shown in WO2012122027.
A detrimental feature of previous instruments that utilize SPIM, including the conversion shown in WO2012122027 is the unconventional positioning of objectives that leads to two key disadvantages. First, sample preparation, sample loading, and sample unloading is cumbersome and challenging. Previous attempts have not been amenable to adapting conventional instruments to maintain the imaging functionality of other powerful microscopy imaging modes, including, but not limited to, SIM (structured illumination microscopy) and TIRF (Total Internal Reflection Fluorescence Microscopy). One way to do this is to rearrange the optic axes of the illumination and imaging objectives. One attempt at such a rearrangement is shown in U.S. Pat. No. 8,472,113. However, the arrangements shown in U.S. Pat. No. 8,472,113 and WO2012122027 use illumination from above, which hinders sample loading and unloading because handling space is consequently very confined.